JPH11507336A - Separation of unmodified hemoglobin from crosslinked hemoglobin - Google Patents

Abstract

  (57) [Summary] A method for separating unmodified hemoglobin from crosslinked hemoglobin in a hemoglobin solution. The method includes contacting the hemoglobin solution with at least one dissociating agent to prepare a dissociation solution in which unmodified tetrameric hemoglobin dissociates to form a hemoglobin dimer. Next, a hemoglobin dimer is separated from the dissociation solution while keeping the crosslinked hemoglobin in the dissociation solution.

Description

DETAILED DESCRIPTION OF THE INVENTION              Separation of unmodified hemoglobin from crosslinked hemoglobin Background of the Invention   Anemia (eg, from acute blood loss or during surgery) resulting from blood loss For example, caused by pernicious anemia or sickle cell anemia) or shock (eg, For example, volume-deficient shock, anaphylactic shock, septic shock or To treat or prevent hypoxia caused by lug shock) Blood is needed.   The use of blood and blood fractions as blood substitutes for these roles has disadvantages. Is accompanied. For example, the use of whole blood often results in hepatitis producing virus and AID With the risk of transmission of the S-producing virus, it is the complete recovery of the patient But can also kill patients. In addition, the use of whole blood, immune blood Blood type and cross-over tests are needed to avoid biological problems and incompatibility between donors. I do.   Hemoglobin as a blood substitute has osmotic activity and the ability to transport and transfer oxygen. Have power. However, water-soluble hemoglobin is a tetramer (molecular weight 68, 000) and dimer (molecular weight 34,000). F Moglobin dimers are excreted by the kidneys and generally have a plasma half-life of 2 to 4 hours. Having such a solution results in acute intravascular elimination of hemoglobin solution.   Modifies hemoglobin molecularly to limit the unique limitations of hemoglobin solutions Efforts have been directed at overcoming them. Intra- and intermolecular hemoglobin Cross-linking generally reduces kidney clearance and increases intravascular maintenance time.   However, in general, solutions of cross-linked hemoglobin are quite crude. The decorated tetrameric hemoglobin fraction is still included. This unmodified tetrameric hemoglobin Can be converted to dimeric hemoglobin and then excreted from the body, Decrease mean intravascular maintenance time for robin blood substitutes. In addition, standard Known methods for separation, such as filtration, include unmodified tetrameric hemoglobin and modified tetramer Hemoglobin cannot be distinguished sufficiently.   Therefore, despite recent advances in the preparation of cross-linked hemoglobin blood substitutes Improves mean intravascular retention time of blood substitutes, and significant levels of hemoglobin in kidney Intramolecular and / or intermolecularly crosslinked hemoglobin charges to prevent elimination There remains a need for an effective method for separating unmodified hemoglobin from blood solutions And continue to exist.Summary of the Invention   The present invention provides an unmodified hemoglobin from a crosslinked hemoglobin in a hemoglobin solution. It relates to a method of separating. The method comprises at least one dissociation a gent) and hemoglobin solution, and unmodified tetrameric hemoglobin is dissociated, Obtaining a dissociation solution in which hemoglobin dimers are formed including. Next, while maintaining the cross-linked hemoglobin in the dissociation solution, The bin dimer is separated from the dissociation solution.   Advantages of the present invention include improved intravascular maintenance time, significant amounts of hemoglobin Reduce or eliminate renal excretion and related side effects, adequate inflation pressure, and high Providing a blood substitute that has a reduced blood pressure effect.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the separation of unmodified hemoglobin from modified hemoglobin blood substitute according to the present invention. 3 shows a schematic flow chart of a release method.   FIG. 2 shows the modified hemoglobin weight in solution, treated by the method of the invention. It shows the molecular weight distribution of the coalescence.Detailed description of the invention   Hb suitable for the hemoglobin (Hb) solution of the present invention is human, bovine, porcine and ovine. Obtained from new, old or old blood from other vertebrates, such as chickens and chickens Can be. Furthermore, the transgenics described in BIO / TECHNOLOGY, 12: 55-59 (1994). Transgenically produced hemoglobin, such as transgenically produced Hb, And Nature, 356: 258-260 (1992). Recombinantly produced hemoglobin is also suitable for the Hb solution of the present invention.   Blood can be collected from live or freshly killed donors. Red blood cells An example of a suitable method for obtaining hemoglobin from is described in Rausch et al. U.S. Pat. Nos. 5,084,558 and 5,296,46 issued to No. 5 is described.   The preferred method of washing and lysing hemoglobin as known in the art From erythrocytes or recombinant bacteria.   A suitable hemoglobin solution comprises an aqueous solution of dissociated Hb, wherein the dissociated Hb is It contains unmodified Hb in addition to the monomeric Hb and / or polymerized Hb.   As defined herein, unmodified hemoglobin dissociates in aqueous solution and b Hemoglobin in an undissociated tetramer that can become a dimer It is an isolated Hb dimer. Furthermore, the Hb dimer is dissociated and the Hb subunit (monomer ) Can be. Unmodified Hb free in Hb solution (not polymerized) And / or cross-linked between molecules in the Hb solution to form polymer chains. You may.   As defined herein, a crosslinked hemoglobin may be modified and / or polymerized. It is what is done. Unmodified Hb contained in the Hb polymer chain is crosslinked between molecules. Any unreacted dimer can be dissociated and separated by the method of the present invention. .   As defined herein, modified hemoglobin is used in aqueous solutions of Hb tetramers. Hb that is intramolecularly crosslinked to prevent significant dissociation of Hb into Hb dimer.   In polymerized hemoglobin, the Hb tetramer is intermolecularly crosslinked to form the Hb polymer chain. Form. Hemoglobin polymers include modified hemoglobin, unmodified hemoglobin, Can include combinations thereof. In this method, if the Hb dimer Does not intermolecularly bind to other Hb tetramers, in aqueous solution, the polymer chains The Hb dimer can be dissociated from the unmodified Hb tetramer within.   In the method of the present invention, at least one dissociating agent is added to hemoglobin in an aqueous solution. To prepare a dissociation solution. A suitable dissociating agent can convert it to unmodified hemoglobin Less hydrogen bonds between Hb dimers in unmodified Hb tetramers when exposed to tetramers Both partially destroyed, dissociated the unmodified Hb tetramer,₁β_TwoAnd / or α_Two β_TwoIt is a water-soluble drug at a concentration in an aqueous solution that produces Hb dimer. You. In addition, the Hb dimer is further dissociated and the Hb subunit (α₁, Α_Two, Β₁as well as β_Two) May be formed.   The dissociation solution is generally about 1 gm / L of the dissociation solution dissolved at a certain concentration. Or more dissociating agents having a normality of the dissociating agent or more. No. Preferably, the dissociator concentration in the dissociation solution is greater than about 1.4N.   The dissociating agent must be ionic or strongly polarized in aqueous solution. Suitable Examples of such dissociating agents include water-soluble inorganic salts (eg, sodium salt, calcium salt). Salts, magnesium salts and zinc salts), water-soluble organic salts (eg, triethylamine Hydrochloride), and water-soluble organic amines (eg, guanidine). Like As a new dissociating agent, an inorganic salt or Ca⁺², Mg⁺²Or Zn⁺²Polyvalent metals like A salt containing at least one cation.   Water solubility, as defined herein, means that the substance is fully soluble in water at room temperature And when contacted with Hb, hydrogen bonds between the Hb dimers in the unmodified Hb tetramer That a solution of sufficient concentration to destroy at least a portion of the means.   The dissociating agent may be dissolved in the aqueous solution before contacting with the Hb solution, or conversely, The dissociating agent is in powder form so that it dissolves subsequently after contact with aqueous Hb solution Alternatively, it may be a particulate solid.   Add either dissociator or Hb solution to the other, or add them together can do. Mix the dissociator and Hb solution under relatively low shear conditions.   Examples of suitable hemoglobin solutions include Bonhard's published The stabilized 2,3-diphosphog described in U.S. Pat. No. 3,864,478. Dissociation agent having a lysate level; Mazur issued U.S. Patent No. 3 No., 925, 344, or the issued rice of Bonsen et al. Japanese Patent Nos. 4,001,200, 4,001,401 and 4 U.S. Pat. No. 5,053,590, or Morris et al., Issued US Published US Patent No. 4,061,736 or Scanon Crosslinked hemoglobin described in U.S. Pat. No. 4,473,496; oczi), issued U.S. Pat. No. 3,991,181, or Simmons et al. ( Simmonds et al.) Issued U.S. Pat. No. 4,401,652, or U.S. Pat. No. 4,526,715 issued to Kothe et al. US Pat. No. 4, issue of Wong, issued from US Pat. Hemoglobin conjugated to polysaccharides described in EP 064,118; Bonhard et al. (B onhard et al.) in issued U.S. Pat. No. 4,136,093. Hemoglobin condensed with pyridoxal phosphate; Bonhard et al. U.S. Pat. No. 4,336,248 issued to U.S. Pat. Synthetic hemoglobin; U.S. Pat. No. 4,3,387 issued to Ajisaka et al. Hemoglobin covalently bound to inulin as described in JP 77,512; Iwashita U.S. Pat. No. 4,412,989 issued to Iwashita et al. Or issued U.S. Pat. No. 4,670,417 to Iwasaki et al. Or issued U.S. Patent No. 5,234,9 to Nho et al. No. 03 with polyalkylene glycol or polyalkylene oxide Binding hemoglobin or hemoglobin derivatives; Bonhard et al. Pyrogen-free as described in issued U.S. Pat. No. 4,439,357. And plasma-free hemoglobin solutions; U.S. Pat. 73,494, a plasma-free, non-hemoproteinaceous hemoglobin; Modifications described in US Pat. No. 4,529,719 issued to Tye. Crosslinked Plasmaless Hemoglobin; U.S. Patent issued by Bucci et al. No. 4,584,130, plasma-free cross-linked hemoglobin; Warda U.S. Pat. No. 4,598,064 issued to Walder, et al. Α-cross-linked hemoglobin described in U.S. Pat. Aldehydes described in U.S. Pat. No. 4,857,636, issued. Combined hemoglobin; Feller et al. Issued U.S. Pat. Covalently linked to the sulfated glycosaminoglycan described in US Pat. No. 4,920,194. US Pat. No. 4,900,78 issued to Cerny. No. 0, which reacts with a high molecular weight polymer having a reactive aldehyde component. Decorated hemoglobin; issued to Hai et al., US Pat. No. 5,128,4. Crosslinked hemoglobin in the presence of sodium tripolyphosphate as described in JP 52 US Patent No. 5,189,149 issued to Hsia; Stable, polyaldehyde-polymerized hemoglobin; developed by Kluger et al. Β-crosslinked hemoglobin described in issued U.S. Pat. No. 5,250,665. Bottles and the like. Other examples of suitable Hb solutions are described in Rausch et al. . U.S. Pat. No. 5,296,465, issued Aug. 19, 1988, which is hereby incorporated by reference.   In a preferred embodiment, the hemoglobin used in the method of the invention is polymerized hemoglobin. It is a form of blood that substitutes for globin. A suitable example of a polymerized hemoglobin blood substitute is U.S. Pat. No. 5,084,558 issued to Rausch et al. And 5,217,648.   A preferred Hb blood substitute composition for use in the method of the invention is 0.5 E Less than dotoxin units / mL, causing no significant decrease in oxygen transport / transfer capacity Methemoglobin content, between about 1 and about 25 g Hb / dl total hemoglobin Sterile aqueous solution with concentration, physiological pH and chloride ion concentration less than 35 meq / L It is.   The term "endotoxin" is produced as an outer membrane part of the bacterial cell wall Exhibits cell-conjugated lipopolysaccharide, which is toxic under many conditions. Endotoxin The unit (EU) is the United States Pharmacopeial Conve ntion, p. 3013, 0.1 nanogram of U.S. reference stan. dard) Defined as activity contained in lot EC-5. One vial of EC-5 Includes 10,000 EU.   Conditions in the dissociation solution, such as pH and temperature, are It is within a range that does not seriously reduce the oxygen carrying and releasing capacity. Such appropriate conditions are Has long been known in the art.   The pH of the dissociation solution was acid-induced, low enough for Hb tetramer dissociation to occur It must be high enough to greatly prevent Hb denaturation. Typically, pH Is maintained between about 4.5 and about 9.5. The pH of the dissociation solution may be acidic preferable.   Also, in other embodiments, within a stable pH range, typically from about 4.5 to about 9.5. The dissociation solution contains a buffer to maintain the dissociation solution. One or more buffers It can consist of a compound.   Preferred buffers include 2,2-bis having a pH range between 5.5 and 8.0. (Hydroxymethyl) -2,2 ', 2' '-nitrilotriethanol (bis-tri S).   Add a solid (powder or particulate) buffer or buffer aqueous solution to the Hb solution. This allows the dissociation solution to be buffered. Further, the buffer is mixed with the Hb solution. Prior to contacting, it can be added to an aqueous solution of the dissociating agent.   In yet another embodiment, the dissociation solution is in the form of methemoglobin by autoxidation. It further contains a suitable amount of stabilizer to minimize formation. Proper amount of stabilizer An example is a 0.1 mM solution of ethylenediaminetetraacetic acid (EDTA).   The hemoglobin solution used in this method is typically used for microbial growth or biological burden (b ioburden) under conditions that can be minimized (e.g., Maintain above 0 ° C and below about 20 ° C). Keep the temperature below about 15 ° C preferable. More preferably, the temperature is maintained between about 10C and about 12C.   Next, the dissociated solution is filtered to dissociate Hb dimer from modified Hb and / or polymerized Hb. To purify the Hb solution. Suitable filters include those in the filtrate And cutout molecules between about 40,000 Daltons and 100,000 Daltons Ultrafiltration devices that pass quantities of components are included. During filtration, the diameter is larger than the modified tetramer Hb The components of the small or fluid or dissolved Hb solution are filtered along with the filtrate. Pass through Luther. However, modified Hb tetramers and polymerized Hb are generally Remains in the retentate.   The 50,000 dalton ultrafiltration device is capable of producing modified Hb tetramers or polymerized Hb. Hb dimer can be separated from Hb solution without significant loss of It is preferred.   In one embodiment, the dissociation solution is filtered only once. Instead, the previous The concentrate from the filter is further purified by the next filter, Two filters allow the dissociation solution to be purified.   The Hb concentrate is continuously recycled through one or more filters as shown in FIG. Preferably during which time unmodified Hb dissociates into the dissociation solution. Continue removing dimers as you continue to do so.   In other embodiments, prior to and / or during filtration, the water or electrolyte Preferably, an aqueous solution of the dissociation agent is added to the dissociation solution, at least individually during filtration. To make up the amount that has flowed off as a filtrate. Water or aqueous solution in batch mode or Add continuously, at the same rate as the rate of filtrate loss through the filter.   The water used in the method of the present invention is distilled water, deionized water, distilled water for injection (WFI). And / or low heating substance water (LPW). WFI relates to distilled water for injection Deionized steam found in the US Pharmacologic Specifications It is preferably distilled water. WFI is Pharmaceutical Engineering, 11, 15-2 3 (1991). LPW contains less than 0.002 EU / mL More preferably, it is ion water.   Typically, the volume of the filtrate removed from the Hb solution before adding the dissociating agent At a time equal to about 500% of the volume of the Hb solution, the modified Hb and the weight in the Hb concentrate About 99% or more of the unmodified Hb is separated from the combined Hb.   Next, when using the Hb solution as a blood substitute, the hemoglobin in the Hb concentrate was removed. Washing and diafiltration with physiological buffer More equilibrate to ensure the physiological tolerance of the blood substitute. Appropriate physiological buffer Include physiologically acceptable levels of electrolytes (eg, NaCl, KCl and CaCl_Two). Buffer is 10,000 It is depyrogenated by filtration using a Dalton ultrafiltration device and deoxygenated. Is preferred.   Further, as the buffer solution, N-acetyl-L-cysteine, cysteine, Contains soluble non-toxic reducing agents such as sodium dithionate or ascorbate Can be used to chemically remove oxygen from blood substitutes Reduce bin formation. The methemoglobin content of the Hb blood substitute is about 2 Above 5% typically results in a significant reduction in oxygen carrying capacity. Met Preferably, the hemoglobin content is less than about 15%. Hb substitute blood More preferably, the tomoglobin content is about 10% or less.   Also, similar to the dissociation buffer, as shown in Example III, Dissociation of unmodified hemoglobin into Hb dimer occurs. However, the oxygen of Hb The metabolism also promotes methemoglobin formation.   Physiological buffer and reducing agent alone or in combination, in batch or continuous It is understood that the feed can be added to the Hb concentrate.   In a preferred embodiment, the Hb solution is physiologically acceptable to humans and / or other vertebrates. By diafiltration against a physiological buffer until Wash the Hb concentrate.   Typically, diafiltration through a diafilter The amount of liquid lost during the washing is about 5 times or more than the initial amount of Hb concentrate before washing. Continue diafiltration until In a more preferred embodiment, Continue diafiltration until about 10 times the liquid volume is changed.   Further details on the use of this method include removing unmodified Hb from a polymer Hb solution. For this purpose, examples I and II are provided.   In this method, there is provided a process for preparing a stable polymerized hemoglobin blood substitute. The components are sanitized sufficiently to produce a sterile product. Sterilization Specifically, as defined in the technical field of USPXXII, Of the United States Pharmacopoeia for sterility as specified in Section 71, pages 1483-1488. Refers to the criteria. In addition, configurations that are exposed to a process stream Always process or process parts with materials that do not react with or contaminate the process stream. Is usually coated. Such materials include stainless steel and Inconel And other steel alloys.   The pump used in this method can be a peristaltic, membrane, gear, piston or low pump. There is a tally lobe type pump. Membrane pumps at Buffalo Grove, Illinois Available from Brune Lubbem Inc. Suitable rotary -Lobe pumps are available from Albin Pump, Inc. of Atlanta, Georgia. Pump Inc.) Alvin SLP 110 P51 B1 sanitary lobe rotary pump Pumps. Rotary lobe pumps are available from War, Wisconsin. Can be obtained from Waukesha Pumps at Key Show .   In one embodiment of system 10, Hb containing modified Hb tetramer and / or polymer Hb Figures that are suitable for performing the method for separating unmodified hemoglobin from solution I. The system 10 includes a tank 12, a pump 14, a purification filter 16, and a die. It has an A-filter 18. The pump 14 applies a suction force on the tank 12 to purify Recirculate the Hb solution through filter 16 and / or diafilter 18 You. The purification filter 16 and the diafilter 18 may be arranged in a parallel or continuous arrangement. Can be activated. Tank 12 can be prepared in tank 12 or tank 12 containing Hb solution that can be prepared before being transferred.   Then, to prepare a dissociation solution, the Hb tetramer is dissociated to Hb dimer. An appropriate amount of the Hb dissociating agent is contacted with the Hb solution in system 10. Typically The dissociation agent is introduced into the tank 12 from the dissociation agent supply device 20. However, the solution It includes that the release agent can be added at other locations in the system 10. Also, batch release agent It also includes that it can be added to the Hb solution by a system or a continuous supply system.   Then, the dissociating agent and the Hb solution in the dissociation solution are mixed with a low-shear mixer, especially a static mixer. Mix by 22. The dissociator and Hb solution were pumped through a hole (not shown). And by recirculating the dissociated solution from tank 12 by static mixer 22. Combine.   The static mixer 22 is typically located downstream of the pump 14 and upstream of the purification filter 16. , But the static mixer 22 is instead located elsewhere in the system 10 be able to.   Then, unmodified hemoglobin in the dissociation solution is converted from unmodified Hb tetramer to Hb dimer. Begin dissociation into the body. Hb dimers each have a molecular weight of about 32,000 daltons have.   The dissociated solution is then recirculated through the purification filter 16 and the Hb The monomer is removed and the modified Hb and the polymer Hb are retained in the Hb concentrate. During filtration First, components of the dissociation solution having a smaller diameter or a fluid than the stabilized tetramer Hb are filtered. The solution is passed through a purification filter 16. An example of a suitable purification filter is about 4 Has a molecular weight cut-out between 000 daltons to about 100,000 daltons Ultrafiltration apparatus.   In continuous feed mode, the rate of filtrate loss through purification filter 16 is the same At a rate, liquid or dissolved dissociation agent is added continuously to make up. In another aspect In this case, the volume of the filtrate discharged through the purification filter 16 is adjusted by a filtration pump 24. Adjust with.   Typically, the volume of filtrate drained from the purification filter 16 contains a dissociating agent in the system 10. Before adding, it is equal to about 500% of the volume of Hb solution contained in tank 22 At times, the separation of unmodified Hb from the Hb concentrate is completed.   Then, in another embodiment, the Hb concentrate is washed and the Hb concentrate is replaced with a blood substitute. Diafiltration with physiological buffer to achieve physiologically acceptable Equilibrate the Hb concentrate. Physiological buffer, physiological buffer supply From the machine 26 into the tank 12. However, a physiological buffer was added to system 10 throat. And that it can be added even at the position. Also, use physiological buffer It also includes that it can be added to the Hb concentrate in a formula or continuous feed mode.   Preferred physiological buffers include 27 mM sodium lactate in WFI, 12 mM M N-acetyl-L-cysteine, 115 mM NaCl and 1.36 mM CaCl_Two(PH 8).   The pump 14 then passes through a static mixer 22 and a diafilter 18. Thus, by recirculating the Hb concentrate and the physiological buffer from the tank 12, b Diafilter the concentrate. The diafilter 18 is a stationary mixer 2 2 and upstream of the tank 12. So that blood substitutes are physiologically acceptable Continue diafiltration until it is. Typically, the diafilter 18 The diafiltration through When at least five times the initial volume of the concentrate, the blood substitute is physiologically acceptable. Become so.   In tank 14 during Hb dissociation and Hb concentrate diafiltration Maintain the Hb temperature at about 8-12C. As an example of good means to control Hb temperature The tank 14 through the use of an ethylene glycol-coated cooling system (not shown). To cool the outside of the car.   The present invention will be further described, but the present invention is not limited by the following examples. Not.                                 Example I Diafiltration of deoxygenated Hb solution containing higher concentration of dissociation buffer   U.S. Pat. No. 5,084,558 issued to Rausch et al. A polymerized Hb solution was prepared according to the method described in Example 1 of the specification. This Hb solution Was analyzed by gel permeation chromatography (GPC). The solution is about 45% Hb dimer, about 15% unmodified Hb tetramer, and unmodified tetramer It was found to consist of about 40% of the larger polymerized Hb molecules.   1.5M MgCl_Two, 0.1 M Bis-Tris and 0.2 mM EDTA ( One liter of the dissociation buffer containing pH 6.5) was added to one liter of the Hb solution. Then this mixing The product was subjected to a 100 kD polysulfone ultrafiltration device (Millipore Catalog No .: PTHK0 Recycle through 00C5) and concentrate the mixture to a volume of 1 L. Next, 0.7 M MgCl_Two, 0.05 M Bis-Tris and 0.1 mM EDTA (pH 6. The concentrated mixture is diafiltrated using 1 liter of dissociation buffer consisting of 5). Torration. Next, the filtered Hb solution was washed, and 27 mM lactic acid was added in WFI. Sodium, 12 mM N-acetylcysteine, 115 mM NaCl, 4 m M KCl and 1.36 mM CaCl_TwoEquilibration using deoxygenation buffer containing did. Next, the molecular weight distribution of the obtained Hb solution was analyzed by GPC.   The results of these analyzes are shown in Figure II. Next, the Hb solution is about 5% Hb dimer, From about 10% Hb tetramer and about 85% polymerized Hb molecules larger than the tetramer It turned out that it was composed.                                 Example II Diafiltration of deoxygenated Hb solution containing lower concentration of dissociation buffer   0.75M MgCl_Two, 0.05 M Bis-Tris and 0.1 mM EDTA 170 mL of the dissociation buffer containing (pH 7.5) was added to the initially polymerized Hb solution 1 of Example I. Added to 5 mL, then added 15 mL of 2x dissociation buffer. Next, Hb The solution is recirculated through a Keminiar / Kenix static mixer, then 100 kD Die using an ultrafiltration device (Amicon YM 100, Catalog No. 14451) Filtration was performed to obtain 200 mL of a Hb solution.   Next, the Hb solution was diafiltered by changing the volume of the dissociation buffer three times. Finally, 27 mM sodium lactate and 12 mM N-acetyl Stain, 115 mM NaCl, 4 mM KCl and 1.36 mM CaCl_Two Washed with deoxygenation buffer containing and equilibrated. Molecular weight distribution of the resulting Hb solution Was analyzed by GPC.   The results of these analyzes are shown in Figure II. Next, the Hb solution is about 15% Hb dimer From about 15% Hb tetramer and about 70% polymerized Hb molecules larger than tetramer. It turned out to be composed eventually. Next, the method of the present invention comprises the steps of: It had the effect of reducing the unmodified hemoglobin content.                                 Example III           Synthesis of oxygenated and deoxygenated Hb solution without dissociation buffer                     And DiafiltrationN   As described in US Pat. No. 5,296,465, bovine whole blood The sample was collected and mixed with the anticoagulant sodium citrate to obtain a blood solution. Was analyzed for endotoxin levels.   After collecting each blood solution sample, maintain at about 2 ° C, then 600 mesh Filtered through a sieve to remove large aggregates and particles.   Before pooling, the penicillin level of each blood solution sample Using a measurement kit purchased from Difco, Detroit, Michigan, `` Rapid Detection of Penicillin in Milk '' The blood solution sample has a penicillin level of 0.00 It was confirmed that it was 8 units / mL or less.   The blood solution samples were then pooled and depyrogenated. ) Mix with aqueous sodium citrate to give 0.2% by weight citric acid in bovine whole blood. A solution containing sodium citrate was obtained (hereinafter referred to as "0.2% sodium citrate blood solution"). )).   The 0.2% sodium citrate blood solution was then applied to 800 μm and 50 μm Continuously passed through a polypropylene filter, a large diameter of about 50μm or more The residue of the blood solution was removed.   The RBC is then washed to remove extracellular plasma proteins, such as BS, from the RBC. A or IgG was separated. To clean the RBCs in the blood solution, First, add the diafiltration tank to the diafiltration tank Was diluted by adding the same amount of filtered isotonic solution as the blood solution in the above. Isotonic solution is Millipo (Catalog No. CDUF 050 G1) 10,000 dalton ultrafiltration membrane Filtered. The isotonic solution was diluted with 6.0 g / L citric acid in distilled water for injection (WFI). Sodium acid, 8.0 g / L sodium chloride. Next Then, the diluted blood solution was added to a 0.2 μm hollow fiber (microgonk). Rosflo II microfiltration cartridge (Microgon Krosfl o Diafilter using II microfiltration cartridge)) To a volume of. At the same time, as a refill, 0.2 μm diafilter The filtered isotonic solution was added continuously at the same rate as the rate of decrease by filtration. Da During the filtration, the diluted blood solution was apparently more direct than the RBCs. For components with small diameters or liquid components such as plasma, a 0.2 μm I passed the filter wall. Larger of RBC, platelets and diluted blood solution Lumps, e.g., leukocytes, survive continuous addition of isotonic solution and become transparent. An analyzed blood solution was obtained.   During the RBC wash, the diluted blood solution is brought to a temperature of about The hydraulic pressure at the filter inlet is maintained at about 25-30 psi to improve the process efficiency. Was.   The volume of the effluent filtered by the diafilter is not diluted with the filtered isotonic solution. When the volume of the blood solution became equal to about 600%, the washing of the RBC was completed. .   The dialysed blood solution is then applied to the Sharpless Super Centrifuge. , Model number AS-16, number 28 with ringdam It was fed continuously at a rate of about 4 liters per minute. Centrifuge operation supplies blood solution Simultaneously, RBCs were separated from leukocytes and platelets. RBC is heavy during operation Enough to separate into different RBC phases, while the white blood cells (WBC) and platelet The number of revolutions sufficient to separate the qualitative part into a light WBC phase, specifically, about 15.0 Rotated at 00 rpm. One fraction of RBC phase and one fraction of WBC phase are separated and continuous During operation, it was withdrawn from the centrifuge.   Following separation of the RBCs, the RBCs were dissolved to obtain a hemoglobin-containing solution. R A substantial portion of the BC is mechanically dissolved as the RBC exits the centrifuge. I let you. R at an angle to the RBC phase flow from inside the centrifuge The cell membrane of the RBC ruptured upon impact on the wall of the BC phase discharge line. Thereby Hemoglobin (Hb) was released from the RBC into the RBC phase.   The dissolved RBC phase was then passed through a discharge line to a static mixer (6 elements (eleme). nts) with Kenics (1/2 inch, Chemineer) Was. At the same time as transferring the RBC phase to the static mixer, an equal amount of WFI is also transferred to the static mixer. Injection where the WFI was mixed with the RBC phase. Stationary mixer of RBC phase and WFI The injection rate into each is about 0.25 L / min.   Dissolved RBC colloid was obtained by mixing the RBC phase and WFI in a static mixer. Was. Then, from the static mixer, the dissolved RBC colloid and Hb were converted into non-hemoglobin. Sharpless Super Centrifuge suitable for separation from RBC components (Model number AS-16, Sharpless Division Alpha-Reyvalce (Alfa-Laval Separation, Inc.). this The centrifuge is sufficient to separate the dissolved RBC colloid into a light Hb phase and a heavy phase. It was rotated at the rotation speed. The lighter phase is approximately equal to or less than Hb and the density of Hb. It was composed of non-hemoglobin components.   From the centrifuge, remove the 0.45 μm Millipore Pellicon cassette (Millipore P ellicon Cassette), Catalog Number HVLP 000 C5 Microfilter Hb phase is continuously withdrawn through the 、, and transferred to a holding tank prepared for Hb purification. Moved. The cell stroma is then microfiltered with the retentate. -Returned to the holding tank. Keep the temperature of the holding tank below 10 ° C during microfiltration. Maintained. In order to improve efficiency, the hydraulic pressure at the inlet of the microfilter is about 10 Microfiltration was terminated when the pressure increased to about 25 psi. Then, microfiltration The performed Hb was transferred from the microfilter to the microfiltration tank.   Then, Hb that had been subjected to microfiltration was subjected to 100,000 millipore catalog number.   Feeded to CDUF 050 H1 ultrafiltration membrane. Included in Hb after microfiltration A substantial portion of Hb and water pass through a 100,000 dalton ultrafiltration membrane And an ultrafiltrate of Hb is obtained, while the fraction exceeding about 100,000 daltons is obtained. Larger cell debris, such as protein, remains and is re-used in the microfiltration tank. Circulated and returned. At the same time, as replenishment, in response to the reduction of water associated with ultrafiltration , WFI were added continuously. Broadly speaking, cell debris is smaller than Hb, All complete, except cellular proteins, electrolytes, coenzymes and metabolic intermediates Contains cellular components and fragments thereof. The Hb concentration in the microfiltration tank is 8 grams / liter. Ultrafiltration was continued until it was less than torr (g / l). Precision for ultrafiltration of Hb The temperature in the filtration tank was maintained at about 10 ° C.   The ultrafiltrate of Hb was transferred to an ultrafiltration tank and then the ultrafiltration of Hb 30,000 Dalton Millipore Cat. No. CDUF 050 T1   Recycle to ultrafiltration membranes for smaller cellular components such as electrolytes, coenzymes, Excluding intermediates and proteins less than about 30,000 daltons, , A concentrated Hb solution of about 100 g Hb / liter was obtained.   The concentrated Hb solution was then removed from the ultrafiltration tank by parallel chromatography. Leads to the medium in a feed column (2 feet long, 8 inches inside diameter) Hb was separated by body chromatography. For chromatography columns Is an anion exchange medium suitable for separating Hb and non-hemoglobin proteins Was included. This anion exchange medium was formed from silica gel. this Silica gel is exposed to gamma-glycidoxypropyl silane to activate epoxide groups. Is formed and then C_ThreeH₇(CH_Three) Quaternary ammonium anion exposed to NCl An exchange medium was formed. This method of treating silica gel Journal of Chromatography, 120: 321-333 (1976).   Each column is first chromatographed with a buffer that promotes Hb binding. The fee column was washed and pretreated. Add 4.52 liters of concentrated Hb solution Each of the chromatography columns was injected. After injection of the concentrated Hb solution, In this way, three different buffers are successively introduced to form a chromatography column. Hb for chromatography by washing and applying a pH gradient in the column Eluted through the column. The temperature of each buffer at the time of use is about 12.4 ° C. there were. Before injecting into the chromatography column, buffer Passed through a 0 Dalton ultrafiltration membrane.   This first buffer, 20 mM Tris-hydroxymethyl aminomethane (toluene) Squirrel) (pH about 8.4 to about 9.4) to chromatograph the concentrated Hb solution. The medium was transferred to the medium in the column for binding to bind Hb. A second buffer with a pH of about 8.3 The buffer, a mixture of the first and third buffers, is then added to the contaminating non- The moglobin component is eluted from the chromatography column while Hb is retained. The pH was adjusted to such a value within the chromatography column. In the second buffer Equilibration is performed at a flow rate of about 3.56 liters per minute per column for about 30 minutes. went. The eluate in the second buffer was discarded as waste. Then a third buffer Liquid, 50 mM Tris (pH about 6.5 to about 7.5), for chromatography Hb was eluted from the column.   The Hb eluate was then compared to 0.22 μSartobran Cat. 5232507 Guided to tank through G1PH filter, where Hb elutes Collected things. The first 3-4% of Hb eluate and the last 3-4% of Hb eluate are waste And   Endotoxin in the eluate is less than 0.05 EU / mL and If the buffer was less than 3.3 nmol / mL, the Hb eluate was used subsequently. 9 liters per 60 liter high purity eluate, 100 g Hb / L 1.0 M sodium chloride, 20 mM Tris (pH 8.9) buffer Thus, an Hb solution having an ionic strength of 160 mM is obtained, and the acid of Hb in the Hb solution is obtained. Reduced affinity. Then, an ultrafiltration membrane, specifically 10,000 daltons Millipore Helicon, Catalog No. CDUF050G1 Fill The Hb solution was recirculated at 10 ° C. until the Hb concentration reached 110 g / L. Was concentrated.   Subsequently, Hoechst-Celanese, Cat. No. G-24 0/40) 0.05μm polypropylene microfilter phase transformer By recirculating the Hb solution through the ferment membrane at 12 liters per minute, Liquid pO_TwoBut HbO_TwoUntil the Hb content is reduced to a level of about 10%. The solution is subjected to a deoxygenation treatment to obtain a deoxygenated Hb solution (hereinafter, “deoxygenated-Hb”). Was. At the same time, nitrogen gas was phase transferred at a flow rate of 60 liters per minute. Led to the other side of Mumbren. During the deoxygenation treatment, the temperature of the Hb solution is increased to about 19 ° C. Maintained between about 31 ° C.   During the deoxygenation treatment and throughout the subsequent process, Hb is added to the hypoxic ring. Maintained under ambient conditions to minimize absorption of oxygen by Hb and deoxygenation- Oxidized Hb (oxyhemoglobin or HbO_Two) Is less than about 10% Degree was maintained.   Then 60 liters of deoxygenated-Hb was added to 180 liters of storage buffer, 0 . 2% by weight N-acetylcysteine, 33 mM sodium phosphate buffer (p H7.8), PO_TwoIs less than 50 torr, using an ultrafiltration membrane Diafiltration was performed to obtain oxidatively stable deoxygenated -Hb. Deoxygenation- Prior to Hb mixing, 10,000 Dalton Millipore Helicon, catalog number Generate heat from the storage buffer using the CDUF050G1 pyrogen removal filter. Sexual substances were removed.   Continuous storage buffer at about the same rate as liquid reduction through ultrafiltration membranes The liquid was added. Diafiltration involves diafiltration through an ultrafiltration membrane. The amount of liquid reduction by torsion is about three times the initial amount of deoxygenated Hb Continued until. This material may be stored at this point.   Oxidation stable deoxygenation-WFI deoxygenated before transfer of Hb to polymerization unit Oxidation-stable deoxygenation-Hb acid Was prevented. The amount of WFI added to the polymerization apparatus depends on the weight of oxidatively stable deoxygenated Hb. Amount of Hb solution having a concentration of about 40 g Hb / L when added to the combined reactor Met. The WFI is then recirculated to the polymerizer to reverse the pressurized nitrogen. 0.05μm polypropylene micro filter IS TRANSFER MEMBRANE (Hoechst-Seranese Co., Catalog No. 5PCM -108, 80 square feet) to remove oxygen from the WFI. Fe The flow rates of WFI and nitrogen gas passing through the Approximately 18 to 20 liters per minute and 40 to 60 liters per minute.   PO of WFI of polymerization equipment_TwoBut about 2 torr pO_TwoAfter being reduced to less than heavy Approximately 20 liters of nitrogen flow per minute into the head space of the Was covered with nitrogen. Next, the oxidation stable deoxygenated-Hb is polymerized into a reactor. Moved to   PH of less than about 35 mM, resulting from mixing of Hb solution and WFI Polymerization was performed in 7.8 phosphate buffer, 12 mM.   Then, under heating conditions of 42 ° C., Kenics with 6 elements 1-1 / 2 inch static Oxidatively stable deoxygenation while recirculating the Hb solution through an in-situ mixer (Cheminer) -Hb and N-acetylcysteine are combined with a crosslinking agent glutaraldehyde, specifically 2g of glutaraldehyde / kg of Hb was added for 5 hours. The mixture was mixed to obtain a polymerized Hb (poly (Hb)) solution.   Oxidatively stable deoxygenation-Recirculate Hb and glutaraldehyde through static mixer Glutaraldehyde and oxidatively stable deoxygenation-Hb under turbulent conditions Gently mixes uniformly, thereby deoxidizing with high concentration of glutaraldehyde The likelihood of formation of pockets of elementary-Hb was reduced. With glutaraldehyde Deoxygenated-by gentle and homogeneous mixing with Hb (molecular weight 500,000 Inhibits the formation of high molecular weight poly (Hb) (over daltons) and furthermore during polymerization Faster mixing of glutaraldehyde and deoxygenated-Hb.   In addition, the effect of N-acetylcysteine during the polymerization of Hb is as follows. Crosslinks were clearly formed in the Hb molecules during the polymerization of Hb.   After the polymerization, the temperature of the poly (Hb) solution in the polymerization reactor is lowered to about 15 ° C. The temperature was between about 25 ° C.   The poly (Hb) solution is then recirculated through the ultrafiltration membrane and the poly (Hb) The poly (Hb) solution was concentrated until the concentration increased to about 85 g / L. Preferred extraordinary As a filtration membrane, a 30,000 dalton filter (for example, Millipore Helicopter) , Catalog number CDUF050LT).   The poly (Hb) solution was then washed with 66.75 g of sodium borohydride And then recirculated again through the static mixer. Specifically, 9 0.25 M sodium borohydride solution for each poly (Hb) solution of toluene One liter was added and the flow rate was 0.1-0.12 liters per minute.   Before adding sodium borohydride to the poly (Hb) solution, the poly (Hb) solution The pH of the solution was adjusted to about pH 10 to make it alkaline, and sodium borohydride was added. Um and hydrogen gas generation. About 215 liters of poly (Hb) solution, 12 mM boric acid with a pH of about 10.4 to about 10.6, excluding pyrogens and oxygen Perform diafiltration with sodium buffer to obtain poly (Hb) solution p H was adjusted. From the polymerization reactor, poly (H) was passed through a 30 kD ultrafiltration membrane. b) Diafiltration was performed by recirculating the solution. Diafiltre At the same rate as the rate of liquid reduction through the ultrafiltration membrane. , A sodium borate buffer was added to the poly (Hb) solution. Diafil The amount of liquid loss due to filtration through the ultrafiltration membrane is Diafiltrate until about 3 times the initial volume of poly (Hb) solution in the filter Ration continued.   Following pH adjustment, a sodium borohydride solution was added to the polymerization reactor. Reduces the imine bonds in the poly (Hb) solution to form ketoimine bonds, (Hb) was stabilized. During the addition of sodium borohydride, The poly (Hb) solution in the Continuous recirculation through the Clofilter Phase transfer membrane The dissolved oxygen and hydrogen were removed. By feeding through a static mixer Mixing sodium borohydride and poly (Hb) solution quickly and effectively It also caused a turbulent flow of sodium borohydride, a feed condition. 0.05μ Poly (Hb) solution and nitrogen gas permeating through m-phase transfer membrane Flow rates of about 2.0 to 4.0 liters per minute and about 12 to 18 liters per minute, respectively Met. After completion of the addition of sodium borohydride, the agitator is rotated at about 75 rpm. While continuing the inversion, the reduction in the polymerization reactor continued.   About 1 hour after the addition of sodium borohydride, 30 110 g of stable poly (Hb) solution through ultrafiltration membrane of 000 daltons / L, the stable poly (Hb) solution was recirculated. Filtered to remove oxygen In addition, 27 mM sodium lactate, 12 mM NAC, 115 mM P containing thorium, 4 mM potassium chloride, and 1.36 mM calcium chloride 30,000 dalton ultrafiltration membrane using low H buffer (pH 5.0) Diafiltration of the stable poly (Hb) solution through After that, the pH and the electrolyte of the stable poly (Hb) solution are returned to physiological levels, and stable polymerization is performed. Hb-substitute blood was obtained. Diafiltration is performed by diafiltration through an ultrafiltration membrane. The amount of liquid reduced by filtration is the diafiltration of the concentrated Hb product. It was continued until the amount before the operation became 6 times.   After returning the pH and the electrolyte of the stable poly (Hb) solution to physiological levels, Filter, add oxygen-removed, low pH buffer to the polymerization reactor, The constant-polymerized hemoglobin blood substitute was diluted to a concentration of 5.0 g / deciliter. Next Then, the diluted blood substitute was transferred from the polymerization reactor to a static mixer and a 100,0 Recirculation through a 00 dalton purification filter filters and removes oxygen. 27 mM sodium lactate, 12 mM NAC, 115 mM salt in WFI Contains sodium chloride, 4 mM potassium chloride, and 1.36 mM calcium chloride Diafiltration was performed on the buffer (pH 7.8). Diaf Filtration was performed by GPC modified under dissociation conditions in blood substitutes. Continued until the amount of monomer and unmodified tetramer was less than about 10%.   Purification filters have a restricted permeate line. And was performed under conditions of low transmembrane pressure. Modified tetramer Hb and unmodified tetramer After most of the Hb has been removed, it is passed through a 30,000 dalton ultrafiltration membrane. Recirculation of the blood substitute was continued until the blood concentration reached 130 g / liter. .   The stable blood substitute is then stored in a preferred container with a low oxygen environment and low oxygen leakage. Existed.   Next, this Hb solution was analyzed by GPC and found to be about 3.5% Hb dimer, 31% Unmodified Hb tetramer and about 65.5% of the polymerized Hb fraction larger than the unmodified tetramer. Turned out to be a child.   Co-Oximeter (Cooximeter # 482; Massachusetts) Instrumentation Institute, Lexington, Setts Laboratory)) until 98% oxygenated Hb valves are obtained. A gaseous mixture of oxygen and 2% carbon dioxide through an oxygenation cartridge One liter of the Hb solution was oxygenated.   Next, 27 mM sodium lactate and 12 mM N-acetyl-L-cy Stain, 115 mM NaCl, 4 mM KCl and 1.4 mM CaCl_Two Oxygenation against 100 kD ultrafiltration device using 7 volumes of oxygenation buffer solution containing The Hb solution was diafiltered. Through this process, the Hb solution is dissociated No contact with buffer.   Next, the molecular weight distribution of the obtained Hb solution was analyzed by GPC. The molecular weight distribution is 0.5% dimer and 2.7% unmodified tetramer and about 96.8% unmodified tetramer It was found to be a larger polymerized Hb.   Next, except that the Hb solution is not oxygenated before diafiltration, This entire process was repeated with another sample of the same polymerized Hb solution. Hb obtained The molecular weight distribution of the solution was determined by GPC to be 2.2% dimer, 2.5% unmodified tetramer and Approximately 95.3% of the polymerized Hb molecules were found to be larger than the unmodified tetramer. .   The results of these steps also indicate that the oxygenation of hemoglobin is similar to unmodified Hb tetramer It is shown to promote dissociation from the body to the Hb dimer. However, also oxygen Also promotes the formation of harmful methemoglobin.                                 Example IV Molecular weight analysis   Gel permeation chromatography on hemoglobin solution under dissociation conditions (GPC) to determine the molecular weight. This analysis method depends on the size Hemoglobin polymer separation is based on larger molecules Elutes faster than larger molecules. Hemoglobin compared to protein molecular weight standards It allows the correlation between product molecular weight and elution time.   In this analysis, a representative sample of hemoglobin products was analyzed for molecular weight distribution. Analyzed for The hemoglobin product was converted to 50 mM Bis-Tris (pH 6.5). ), 750 mM MgCl_TwoAnd 4m inside a mobile phase of 0.1mM EDTA g / mL. Dilute the sample with HPLC Toso Haas G3000SW The ram was injected. The flow rate was 0.5 mL / min and UV detection was performed at 280 nm. Was.Equivalent   Those skilled in the art will recognize, by mere routine experimentation, the invention described herein. Many equivalents to a particular embodiment can be recognized or confirmed. Would. These and all other such equivalents fall within the scope of the following claims. It is rare.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] May 15, 1997 [Correction contents] First, a diafiltration tray is used to clean the RBCs contained in the blood solution. Filter the same amount of blood solution as in the diafiltration tank. The diluted isotonic solution was added for dilution. Isotonic solutions are available from Millipore (Cat.   050 G1) Filtered through a 10,000 dalton ultrafiltration membrane. Isotonic solution 6.0 g / L sodium citrate, 8.0 g / L in distilled water for injection (WFI) Of sodium chloride. Then, the diluted blood solution , 0.2μm hollow fiber (Microgon Crossflow II Micro Filtration cartridge (Microgon Krosflo II microfiltration car Concentrated to the original volume by diafilter using tridge)). same Sometimes, as a replenishment, the rate of decrease by filtration through a 0.2 μm diafilter and At the same rate, the filtered isotonic solution was added continuously. Diafiltration In between, components of the diluted blood solution, apparently smaller in diameter than RBC, or plasma The liquid components such as passed through the walls of a 0.2 μm diafilter by filtration. Larger clumps of RBCs, platelets and diluted blood solution, such as leukocytes, A residual and dialyzed blood solution was obtained by continuous isotonic addition.   During the RBC wash, the diluted blood solution is brought to a temperature of about The hydraulic pressure at the inlet of the filter is about 120 N / m^Two(25 psi) to about 140 N / m^Two(3 0 psi) to improve the efficiency of the process.   The volume of the effluent filtered by the diafilter is not diluted with the filtered isotonic solution. When the volume of the blood solution became equal to about 600%, the washing of the RBC was completed. .   The dialysed blood solution is then applied to the Sharpless Super Centrifuge. , Model number AS-16, number 28 with ringdam It was fed continuously at a rate of about 4 liters per minute. Centrifuge operation supplies blood solution Simultaneously, RBCs were separated from leukocytes and platelets. RBC is heavy during operation Enough to separate into different RBC phases, while the white blood cells (WBC) and platelet The number of revolutions sufficient to separate the qualitative part into a light WBC phase, specifically, about 15.0 Rotated at 00 rpm. One fraction of RBC phase and one fraction of WBC phase are separated and continuous During operation, it was withdrawn from the centrifuge.   Following separation of the RBCs, the RBCs were dissolved to obtain a hemoglobin-containing solution. R A substantial portion of the BC is mechanically dissolved as the RBC exits the centrifuge. I let you. R at an angle to the RBC phase flow from inside the centrifuge The cell membrane of the RBC ruptured upon impact on the wall of the BC phase discharge line. Thereby Hemoglobin (Hb) was released from the RBC into the RBC phase.   The dissolved RBC phase was then passed through a discharge line to a static mixer (6 elements (eleme). nts) with Kenics (1/2 inch, Chemineer) Was. At the same time as transferring the RBC phase to the static mixer, an equal amount of WFI is also transferred to the static mixer. Injection where the WFI was mixed with the RBC phase. Stationary mixer of RBC phase and WFI Injection rate to each about 4 × 10^-6m^ThreePer second (0.25 liters per minute) You.   Dissolved RBC colloid was obtained by mixing the RBC phase and WFI in a static mixer. Was. Then, from the static mixer, the dissolved RBC colloid and Hb were converted into non-hemoglobin. Sharpless Super Centrifuge suitable for separation from RBC components (Model number AS-16, Sharpless Division Alpha-Rival) (Alfa-Laval Separation, Inc.). This Enough to separate the dissolved RBC colloid into a light Hb phase and a heavy phase Was rotated at an appropriate rotation speed. The lighter phase is more or less equal to Hb and the density of Hb It was composed of small non-hemoglobin components.   From the centrifuge, remove the 0.45 μm Millipore Pellicon cassette (Millipore P ellicon Cassette), Catalog Number HVLP 000 C5 Microfilter Hb phase is continuously withdrawn through the 、, and transferred to a holding tank prepared for Hb purification. Moved. The cell stroma is then microfiltered with the retentate. -Returned to the holding tank. Keep the temperature of the holding tank below 10 ° C during microfiltration. Maintained. In order to improve efficiency, the hydraulic pressure at the inlet of the microfilter should be about 48 N / m^Two(10 psi) to about 120 N / m^Two(25 psi) when accurate Filtration was completed. Next, the Hb that has been subjected to the microfiltration is filtered through the microfilter. Transferred to overuse tank.   Then, Hb that had been subjected to microfiltration was subjected to 100,000 millipore catalog number.   Feeded to CDUF 050 H1 ultrafiltration membrane. Included in Hb after microfiltration A substantial portion of Hb and water pass through a 100,000 dalton ultrafiltration membrane And an ultrafiltrate of Hb is obtained, while the fraction exceeding about 100,000 daltons is obtained. Larger cell debris, such as protein, remains and is re-used in the microfiltration tank. Circulated and returned. At the same time, as replenishment, in response to the reduction of water associated with ultrafiltration , WFI were added continuously. Broadly speaking, cell debris is smaller than Hb, All complete, except cellular proteins, electrolytes, coenzymes and metabolic intermediates Contains cellular components and fragments thereof. The Hb concentration in the microfiltration tank is 8 grams / liter. Ultrafiltration was continued until it was less than torr (g / l). Precision for ultrafiltration of Hb The temperature in the filtration tank was maintained at about 10 ° C.   The ultrafiltrate of Hb was transferred to an ultrafiltration tank and then the ultrafiltration of Hb 30,000 Dalton Millipore Cat. No. CDUF 050 T1   Recycle to ultrafiltration membranes for smaller cellular components such as electrolytes, coenzymes, Excluding intermediates and proteins less than about 30,000 daltons, , A concentrated Hb solution of about 100 g Hb / liter was obtained.   The concentrated Hb solution was then removed from the ultrafiltration tank by parallel chromatography. Leads to the medium in a feed column (2 feet long, 8 inches inside diameter) Hb was separated by body chromatography. For chromatography columns Is an anion exchange medium suitable for separating Hb and non-hemoglobin proteins Was included. This anion exchange medium was formed from silica gel. this Silica gel is exposed to gamma-glycidoxypropyl silane to activate epoxide groups. Is formed and then C_ThreeH₇(CH_Three) Quaternary ammonium anion exposed to NCl An exchange medium was formed. This method of treating silica gel Journal of Chromatography, 120: 321-333 (1976).   Each column is first chromatographed with a buffer that promotes Hb binding. The fee column was washed and pretreated. Add 4.52 liters of concentrated Hb solution Each of the chromatography columns was injected. After injection of the concentrated Hb solution, In this way, three different buffers are successively introduced to form a chromatography column. Hb for chromatography by washing and applying a pH gradient in the column Eluted through the column. The temperature of each buffer at the time of use is about 12.4 ° C. there were. Before injecting into the chromatography column, buffer Passed through a 0 Dalton ultrafiltration membrane.   This first buffer, 20 mM Tris-hydroxymethyl aminomethane (toluene) Squirrel) (pH about 8.4 to about 9.4) to chromatograph the concentrated Hb solution. The medium was transferred to the medium in the column for binding to bind Hb. A second buffer with a pH of about 8.3 The buffer, a mixture of the first and third buffers, is then added to the contaminating non- The moglobin component is eluted from the chromatography column while Hb is retained. The pH was adjusted to such a value within the chromatography column. In the second buffer The equilibration was about 5.93 × 10 per column.^-Fivem^Three/ Sec (3.56 l / min) At a flow rate of about 30 minutes. The eluate in the second buffer is waste Threw away. Then, a third buffer, 50 mM Tris (pH from about 6.5 to about 7. 5), Hb was eluted from the chromatography column.   The Hb eluate was then compared to 0.22 μSartobran Cat. 5232507 Guided to tank through G1PH filter, where Hb elutes Collected things. The first 3-4% of Hb eluate and the last 3-4% of Hb eluate are waste And   Endotoxin in the eluate is less than 0.05 EU / mL and If the buffer was less than 3.3 nmol / mL, the Hb eluate was used subsequently. 9 liters per 60 liter high purity eluate, 100 g Hb / L 1.0 M sodium chloride, 20 mM Tris (pH 8.9) buffer Thus, an Hb solution having an ionic strength of 160 mM is obtained, and the acid of Hb in the Hb solution is obtained. Reduced affinity. Then, an ultrafiltration membrane, specifically 10,000 daltons Millipore Helicon, Catalog No. CDUF050G1 Fill The Hb solution was recirculated at 10 ° C. until the Hb concentration reached 110 g / L. Was concentrated.   Subsequently, Hoechst-Celanese, Cat. No. G-24 0/40) 0.05μm polypropylene microfilter phase transformer By recirculating the Hb solution through the ferment membrane at 12 liters per minute, Liquid PO_TwoBut HbO_TwoUntil the Hb content is reduced to a level of about 10%. The solution is subjected to a deoxygenation treatment to obtain a deoxygenated Hb solution (hereinafter, “deoxygenated-Hb”). Was. At the same time, nitrogen gas was phase transferred at a flow rate of 60 liters per minute. Led to the other side of Mumbren. During the deoxygenation treatment, the temperature of the Hb solution is increased to about 19 ° C. Maintained between about 31 ° C.   During the deoxygenation treatment and throughout the subsequent process, Hb is added to the hypoxic ring. Maintained under ambient conditions to minimize absorption of oxygen by Hb and deoxygenation- Oxidized Hb (oxyhemoglobin or HbO_Two) Is less than about 10% Degree was maintained.   Then 60 liters of deoxygenated-Hb was added to 180 liters of storage buffer, 0 . 2% by weight N-acetylcysteine, 33 mM sodium phosphate buffer (p H7.8), PO_TwoIs 6.7 × 10^ThreeN / m^Two(50 torr) or less Diafiltration through an ultrafiltration membrane and stable oxidation and deoxygenation -Hb was obtained. Prior to deoxygenation-Hb mixing, 10,000 Dalton Millipore   Helicon, using exothermic substance removal filter of catalog number CDUF050G1 And the pyrogen was removed from the storage buffer.   Continuous storage buffer at about the same rate as liquid reduction through ultrafiltration membranes The liquid was added. Diafiltration involves diafiltration through an ultrafiltration membrane. The amount of liquid reduction by torsion is about three times the initial amount of deoxygenated Hb Continued until. This material may be stored at this point.   Oxidation stable deoxygenation-WFI deoxygenated before transfer of Hb to polymerization unit Oxidation-stable deoxygenation-Hb acid Was prevented. The amount of WFI added to the polymerization apparatus depends on the weight of oxidatively stable deoxygenated Hb. Amount of Hb solution having a concentration of about 40 g Hb / L when added to the combined reactor Met. The WFI is then recirculated to the polymerizer to reverse the pressurized nitrogen. 0.05μm polypropylene micro filter IS TRANSFER MEMBRANE (Hoechst-Seranese Co., Catalog No. 5PCM -108, 7.4m^Two(80 sq. Ft.) Oxygen from WFI by flow through Was removed. Of WFI and nitrogen gas passing through the phase transfer membrane Each flow rate is about 3 × 10^-Four~ 3.3 × 10^-Fourm^Three/ Sec (18-20 liters per minute) Torr), 6.7 × 10^-Four~ 1 × 10^-3m^ThreePer second (40-60 liters per minute) Was.   PO of WFI of polymerization equipment_TwoBut about 2 torr pO_TwoAfter being reduced to less than heavy About 3.33 × 10 in the head space of the combined reactor^-Fourm^Three/ Second (20 liters per minute ) And the polymerization reactor was covered with nitrogen. Then, oxidation stable deoxidation The primed-Hb was transferred to the polymerization reactor.   PH of less than about 35 mM, resulting from mixing of Hb solution and WFI Polymerization was performed in 7.8 phosphate buffer, 12 mM.   Then, under heating conditions of 42 ° C., Kenics with 6 elements 1-1 / 2 inch static Oxidatively stable deoxygenation while recirculating the Hb solution through an in-situ mixer (Cheminer) -Hb and N-acetylcysteine are combined with a crosslinking agent glutaraldehyde, specifically 2g of glutaraldehyde / kg of Hb was added for 5 hours. The mixture was mixed to obtain a polymerized Hb (poly (Hb)) solution.   Oxidatively stable deoxygenation-Recirculate Hb and glutaraldehyde through static mixer Glutaraldehyde and oxidatively stable deoxygenation-Hb under turbulent conditions Gently mixes uniformly, thereby deoxidizing with high concentration of glutaraldehyde The likelihood of formation of pockets of elementary-Hb was reduced. With glutaraldehyde Deoxygenated-by gentle and homogeneous mixing with Hb (molecular weight 500,000 Inhibits the formation of high molecular weight poly (Hb) (over daltons) and furthermore during polymerization Faster mixing of glutaraldehyde and deoxygenated-Hb.   In addition, the effect of N-acetylcysteine during the polymerization of Hb is as follows. Crosslinks were clearly formed in the Hb molecules during the polymerization of Hb.   After the polymerization, the temperature of the poly (Hb) solution in the polymerization reactor is lowered to about 15 ° C. The temperature was between about 25 ° C.   The poly (Hb) solution is then recirculated through the ultrafiltration membrane and the poly (Hb) The poly (Hb) solution was concentrated until the concentration increased to about 85 g / L. Preferred extraordinary As a filtration membrane, a 30,000 dalton filter (for example, Millipore Helicopter) , Catalog number CDUF050LT).   The poly (Hb) solution was then washed with 66.75 g of sodium borohydride And then recirculated again through the static mixer. Specifically, 9 0.25 M sodium borohydride solution for each poly (Hb) solution of toluene One liter was added and the flow rate was 0.1-0.12 liters per minute.   Before adding sodium borohydride to the poly (Hb) solution, the poly (Hb) solution The pH of the solution was adjusted to about pH 10 to make it alkaline, and sodium borohydride was added. Um and hydrogen gas generation. About 215 liters of poly (Hb) solution, 12 mM boric acid with a pH of about 10.4 to about 10.6, excluding pyrogens and oxygen Perform diafiltration with sodium buffer to obtain poly (Hb) solution p H was adjusted. From the polymerization reactor, poly (H) was passed through a 30 kD ultrafiltration membrane. b) Diafiltration was performed by recirculating the solution. Diafiltre At the same rate as the rate of liquid reduction through the ultrafiltration membrane. , A sodium borate buffer was added to the poly (Hb) solution. Diafil The amount of liquid loss due to filtration through the ultrafiltration membrane is Diafiltrate until about 3 times the initial volume of poly (Hb) solution in the filter Ration continued.   Following pH adjustment, a sodium borohydride solution was added to the polymerization reactor. Reduces the imine bonds in the poly (Hb) solution to form ketoimine bonds, (Hb) was stabilized. During the addition of sodium borohydride, The poly (Hb) solution in the Continuous recirculation through the Clofilter Phase transfer membrane The dissolved oxygen and hydrogen were removed. By feeding through a static mixer Mixing sodium borohydride and poly (Hb) solution quickly and effectively It also caused a turbulent flow of sodium borohydride, a feed condition. 0.05μ Poly (Hb) solution and nitrogen gas permeating through m-phase transfer membrane Flow rates of about 3.3 × 10^-Five~ 6.7 × 10^-Fivem^Three/ Second (from 2.0 to 4.0 liters) and about 2 × 10^-Four~ 3 × 10^-Fourm^Three/ Sec (12-18 liters per minute Le). After completion of the addition of sodium borohydride, the stirrer is rotated at about 75 rpm. The reduction in the polymerization reactor continued during the rotation of.   About 1 hour after the addition of sodium borohydride, 30 110 g of stable poly (Hb) solution through ultrafiltration membrane of 000 daltons / L, the stable poly (Hb) solution was recirculated. Filtered to remove oxygen In addition, 27 mM sodium lactate, 12 mM NAC, 115 mM P containing thorium, 4 mM potassium chloride, and 1.36 mM calcium chloride 30,000 dalton ultrafiltration membrane using low H buffer (pH 5.0) Diafiltration of the stable poly (Hb) solution through After that, the pH and the electrolyte of the stable poly (Hb) solution are returned to physiological levels, and stable polymerization is performed. Hb-substitute blood was obtained. Diafiltration is performed by diafiltration through an ultrafiltration membrane. The amount of liquid reduced by filtration is the diafiltration of the concentrated Hb product. It was continued until the amount before the operation became 6 times.   After returning the pH and the electrolyte of the stable poly (Hb) solution to physiological levels, Filter, add oxygen-removed, low pH buffer to the polymerization reactor, The constant-polymerized hemoglobin blood substitute was diluted to a concentration of 5.0 g / deciliter. Next Then, the diluted blood substitute was transferred from the polymerization reactor to a static mixer and a 100,0 Recirculation through a 00 dalton purification filter filters and removes oxygen. 27 mM sodium lactate, 12 mM NAC, 115 mM salt in WFI Contains sodium chloride, 4 mM potassium chloride, and 1.36 mM calcium chloride Diafiltration was performed on the buffer (pH 7.8). Diaf Filtration was performed by GPC modified under dissociation conditions in blood substitutes. Continued until the amount of monomer and unmodified tetramer was less than about 10%.   Purification filters have a restricted permeate line. And was performed under conditions of low transmembrane pressure. Modified tetramer Hb and unmodified tetramer After most of the Hb has been removed, it is passed through a 30,000 dalton ultrafiltration membrane. Recirculation of the blood substitute was continued until the blood concentration reached 130 g / liter. .   The stable blood substitute is then stored in a preferred container with a low oxygen environment and low oxygen leakage. Existed.   Next, this Hb solution was analyzed by GPC and found to be about 3.5% Hb dimer, 31% Unmodified Hb tetramer and about 65.5% of the polymerized Hb fraction larger than the unmodified tetramer. Turned out to be a child.   Co-Oximeter (Cooximeter # 482; Massachusetts) Instrumentation Institute, Lexington, Setts Laboratory)) until 98% oxygenated Hb valves are obtained. A gaseous mixture of oxygen and 2% carbon dioxide through an oxygenation cartridge One liter of the Hb solution was oxygenated.   Next, 27 mM sodium lactate and 12 mM N-acetyl-L-cy Stain, 115 mM NaCl, 4 mM KCl and 1.4 mM CaCl_Two Oxygenation against 100 kD ultrafiltration device using 7 volumes of oxygenation buffer solution containing The Hb solution was diafiltered. Through this process, the Hb solution is dissociated No contact with buffer.   Next, the molecular weight distribution of the obtained Hb solution was analyzed by GPC. The molecular weight distribution is 0.5% dimer and 2.7% unmodified tetramer and about 96.8% unmodified tetramer It was found to be a larger polymerized Hb.   Next, except that the Hb solution is not oxygenated before diafiltration, This entire process was repeated with another sample of the same polymerized Hb solution. Hb obtained The molecular weight distribution of the solution was determined by GPC to be 2.2% dimer, 2.5% unmodified tetramer and Approximately 95.3% of the polymerized Hb molecules were found to be larger than the unmodified tetramer. .   The results of these steps also indicate that the oxygenation of hemoglobin is similar to unmodified Hb tetramer It is shown to promote dissociation from the body to the Hb dimer. However, also oxygen Also promotes the formation of harmful methemoglobin.                                 Example IV Molecular weight analysis   Gel permeation chromatography on hemoglobin solution under dissociation conditions (GPC) to determine the molecular weight. This analysis method depends on the size Hemoglobin polymer separation is based on larger molecules Elutes faster than larger molecules. Hemoglobin compared to protein molecular weight standards It allows the correlation between product molecular weight and elution time.   In this analysis, a representative sample of hemoglobin products was analyzed for molecular weight distribution. Analyzed for The hemoglobin product was converted to 50 mM Bis-Tris (pH 6.5). ), 750 mM MgCl_TwoAnd 4m inside a mobile phase of 0.1mM EDTA g / mL. Dilute the sample with HPLC Toso Haas G3000SW The ram was injected. The flow rate was 0.5 mL / min and UV detection was performed at 280 nm. Was. 9. The method according to claim 1, wherein the dissociating agent is a water-soluble organic amine. 10. The method according to claim 9, wherein the amine is guanidine. 11. 2. The method according to claim 1, further comprising the step of contacting the hemoglobin solution with a buffer. the method of. 12. The Hb solution was diluted with 2,2-bis (hydroxymethyl) -2,2 ', 2' '-nitri The method according to claim 11, wherein the buffering is carried out using rotriethanol. 13. The method of claim 1, further comprising the step of contacting the Hb solution with a stabilizer. . 14． 14. The method according to claim 13, wherein the stabilizer is ethylenediaminetetraacetic acid. 15. Separation of unmodified hemoglobin from dissociated solution by filtering the dissociated solution The method of claim 1, wherein 16. Separation of unmodified hemoglobin from the dissociation solution followed by a physiologically acceptable buffer Wash the crosslinked hemoglobin with a liquid to obtain a physiologically acceptable blood substitute for hemoglobin. The method of claim 1, further comprising the step of producing a liquid. 17． From the cross-linked product in a hemoglobin solution, the method comprises the following steps, A method for separating bottles and producing a physiologically acceptable blood substitute, a) contacting the hemoglobin solution with a dissociating agent to obtain a dissociation solution, A process in which the decorated tetrameric hemoglobin is dissociated to form a hemoglobin dimer, b) While maintaining cross-linked hemoglobin in the concentrate, the hemoglobin Filtering the monomer, and c) washing the cross-linked hemoglobin with a physiological buffer to provide a physiologically acceptable substitute The process of producing blood. 18. A buffer containing sodium lactate and N-acetyl-L-cysteine. 18. The method according to claim 17. 19. Unmodified from cross-linked hemoglobin in hemoglobin solution with the following steps How to separate hemoglobin, a) Mixing hemoglobin solution with dissociator, buffer and stabilizer to prepare dissociation solution In which unmodified hemoglobin dissociates and hemoglobin Producing a dimer, b) filtration of the dissociation solution to separate hemoglobin dimer from cross-linked hemoglobin And e) containing sodium lactate, N-acetyl-L-cysteine and physiological electrolytes Washing the crosslinked hemoglobin solution using a deoxygenated solution.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I L, IS, JP, KE, KG, KP, KR, KZ, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, TJ, TM, TR , TT, UA, UG, UZ, VN (72) Inventors Lasetti, Anthony Jay.             United States Massachusetts             01845 NORTH ANDOVER, CAND             Rustic Road 70 (72) Houtchens, Robert A.             United States Massachusetts             01757 Milford, Briar Dora             Eve 22

Claims (1)

[Claims] 1. A method for separating unmodified hemoglobin from crosslinked hemoglobin in a hemoglobin solution, comprising the steps of: a) contacting a hemoglobin solution with at least one dissociating agent to obtain a dissociation solution, wherein the unmodified tetrameric hemoglobin is obtained. Is dissociated to form a hemoglobin dimer, and b) a step of separating the hemoglobin dimer from the dissociation solution while keeping the crosslinked hemoglobin in the dissociation solution. 2. The method of claim 1, wherein at least a portion of the hemoglobin in the hemoglobin solution is polymerized. 3. The method according to claim 1, wherein the dissociating agent is a water-soluble inorganic salt. 4. 4. The method according to claim 3, wherein the inorganic salt is a sodium salt. 5. 4. The method according to claim 3, wherein the inorganic salt comprises a polyvalent cation. 6. The method of claim 5, wherein the polyvalent cation is selected from the group consisting of Ca ^{+ 2} , Mg ^{+ 2} , Zn ^{+ 2} and combinations thereof. 7. The method according to claim 1, wherein the dissociating agent is a water-soluble organic salt. 8. The method according to claim 7, wherein the organic salt is a salt of an amine. 9. The method according to claim 1, wherein the dissociating agent is a water-soluble organic amine. 10. The method according to claim 9, wherein the amine is guanidine. 11. The method of claim 1, further comprising the step of mixing the dissociating agent and the hemoglobin solution under low shear conditions. 12. The method of claim 1, further comprising the step of contacting the hemoglobin solution with a buffer. 13. 13. The method according to claim 12, wherein the Hb solution is buffered with 2,2-bis (hydroxymethyl) -2,2 ', 2 "-nitrilotriethanol. 14． The method of claim 1, further comprising contacting the Hb solution with a stabilizer. 15. The method according to claim 14, wherein the stabilizer is ethylenediaminetetraacetic acid. 16. The method according to claim 1, wherein unmodified hemoglobin is separated from the dissociated solution by filtering the dissociated solution. 17． The method according to claim 1, further comprising a step of separating the unmodified hemoglobin from the dissociation solution, washing the crosslinked hemoglobin with a physiologically acceptable buffer, and producing a physiologically acceptable hemoglobin blood substitute. 18. A method of separating unmodified hemoglobin from a crosslinked product in a hemoglobin solution and producing a physiologically acceptable blood substitute, comprising the following steps: a) contacting the hemoglobin solution with a dissociating agent to obtain a dissociation solution; Dissociating the unmodified tetrameric hemoglobin to form a hemoglobin dimer; b) filtering the hemoglobin dimer from the dissociated solution while retaining the crosslinked hemoglobin in the concentrate; and c). Washing the crosslinked hemoglobin with a physiological buffer to produce a physiologically acceptable blood substitute. 19. 20. The method of claim 19, wherein the buffer contains sodium lactate and N-acetyl-L-cysteine. 20. A method for separating unmodified hemoglobin from crosslinked hemoglobin in a hemoglobin solution, comprising the steps of: a) mixing a hemoglobin solution with a dissociating agent, a buffer and a stabilizer to prepare a dissociation solution, Mixing by shearing to dissociate unmodified hemoglobin to form a hemoglobin dimer in the dissociation solution; b) filtering the dissociation solution and separating hemoglobin dimer from cross-linked hemoglobin; and e) lactic acid Washing the crosslinked hemoglobin solution with a deoxygenated solution containing sodium, N-acetyl-L-cysteine and a physiological electrolyte.